Mass spectrometry (MS) is a technology that allows the precise determination of the mass of molecules. It is widely used in numerous applications in life- and other sciences and today it is considered to be one of the most relevant analytical platforms in the characterization of proteins and peptides, where it allows generating a holistic picture of many properties of almost all proteins—the proteome—in a cell or tissue. Attempts to globally study all proteins in a biological sample are usually described using the umbrella term proteomics.
There are a number of approaches to use MS to identify, characterize, or quantify proteins, but the most widely applied strategy is the so-called “bottom-up” approach where specific enzymes are used to cleave proteins at well-defined positions to generate peptides, which are then subjected to MS. MS generally only allows the analysis of molecules carrying a charge (i.e., ions) and therefore peptides, prior to being subjected to the mass spectrometer, are usually ionized using one out of several ionization techniques, such as electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), or any other suitable technology.
A common way of processing peptides in the mass spectrometer is to first determine the mass—actually the mass-to-charge ratio (m/z)—of the intact peptide in an MS1 experiment and then generating additional information regarding the structure of the peptide ion in an MS2 experiment by fragmenting the peptide ions into smaller ions followed by the measurement of the m/z values of these so-called fragment ions. Usually, the collected information used in combination with protein sequence databases of the studied organisms is sufficient to obtain the amino acid sequence of the analyzed peptides, which allows one to infer information about the proteins of the studied sample.
When a proteomics experiment, which often analyzes 10,000 s of peptides in a single experiment, is performed to obtain quantitative information, the experiment most frequently results in relative quantitative data by comparing two or more specific samples. Peptides from each sample may be derivatized or labeled with certain stable isotopes (e.g. carbon-13 or nitrogen-15), so that after pooling the samples, an identical but differentially labeled pair of peptides can be distinguished in the mass spectrometer and the measured peptide ion intensity may be used to obtain accurate quantitative information about concentration differences of this peptide between the studied samples. One shortcoming of mass spectrometry-based proteomics experiments is that they require relatively long acquisition times on rather expensive mass spectrometers. Accordingly, there is considerable effort put into the development of methods that allow multiplexed quantitative experiments—the parallel quantitative comparison of several samples in just one experiment. The development of specially designed chemical tags, such as tandem mass tags (TMTs) and isobaric tags for relative and absolute quantitation (iTRAQ), has provided the ability to perform multiplexed quantitation of a plurality of samples simultaneously. Performing a multiplexed quantitation allows the relative quantities of particular proteins or peptides between samples to be determined. For example, multiplexed quantitation may be used to identify differences between two tissue samples, which may comprise thousands of unique proteins.
The chemical tags are included in reagents used to treat peptides as part of sample processing. A different tag may be used to label each separate sample. Each of the plurality of tags may be isobaric, meaning each of the types of tags has nominally the same mass and are therefore indistinguishable in an MS1 spectrum. This is achieved by using different isotopes of the same elements in the creation of the tags. For example, a first tag may use a carbon-12 atom at a particular location of the molecule, whereas as second tag may use a carbon-13 atom—resulting in a weight difference of approximately one Dalton at that particular location. This purposeful selection of particular isotopes may be done at a plurality of locations for a plurality of elements. As a whole, each isotope of each tag is selected so that the different types of tags have the same total mass resulting in tagged precursor ions with nominally the same mass despite being labeled with a different type of tag. The different isotopes are strategically distributed within the tag molecule such that, when the tag is fragmented, the portion of the tag molecule that will become a low-mass reporter ion for each type of tag has a different weight. Thus, when the different types of tags are fragmented during the MS2 analysis techniques, each type of tag will yield reporter ions with distinguishable mass-to-charge (m/z) ratios. The intensity of the reporter ion signal for a given tag is indicative of the amount of the tagged protein or peptide within the sample. Accordingly, multiple samples may be tagged with different tags and simultaneously analyzed to directly compare the difference in the quantity of particular proteins, peptides or molecules in each sample.